Particle-mediated bombardment of DNA sequences into tissue to induce an immune response

ABSTRACT

A method of transferring a gene to vertebrate cells is disclosed. The method comprises the steps of: (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the tissue cells and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and (b) accelerating the microprojectiles at the cells, with the microprojectiles contacting the cells at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein. Preferably, the target cells reside in situ in the animal subject when they are transformed. Preferred target cells are dermis or hypodermis cells, and preferred genes for insertion into the target cells are genes which code for proteins or peptides which produce a physiological response in the animal subject.

This application is a continuation of application Ser. No. 08/103,814,filed Aug. 6, 1993 (now abandoned); which is a continuation of Ser. No.07/864,638, filed Apr. 7, 1992 (now abandoned); which is a divisional ofSer. No. 07/437,848, filed Nov. 16, 1989 (now abandoned.)

SUMMARY OF THE INVENTION

This invention relates to the transformation of animal cells and tissuewith heterologous DNA by microprojectile bombardment.

BACKGROUND OF THE INVENTION

The transformation of living cells by propelling microprojectiles atthose cells at high velocity, with the microprojectiles carryingexogenous DNA or RNA, was originally proposed by T. Klein, E. Wolf, R.Wu and J. Sanford, Nature 327, 70 (1987). See also J. Sanford et al.,Particulate Science and Technology 5, 27 (1987). The original workinvolved the tranformation of onion epidermal cells with RNA derivedfrom tobacco mosaic virus. The findings with onion epidermal cells havebeen extended to other plants. For example, the transformation ofsoybean callus by particle bombardment is described by P. Christou etal., Plant Physiol. 87, 671 (1988), and the transformation of soybeanmeristem is described by D. McCabe et al., Bio/Technology 6, 923 (1988).European Patent Application Publication No. 0 301 749 to P. Christou etal. The transformation of embryonic maize callus cells by particlebombardment is described by T. Klein et al., Proc. Natl. Acad. Sci. USA85, 4305 (1988), and the production of transformed maize seed by theparticle bombardment of maize pollen is described in European PatentApplication Publication No. 0 270 357 to D. McCabe et al.

In addition to the transformation of plants, microprojectile bombardmenthas been used to transform cellular organelles. Mitochondrialtransformation in yeast by particle bombardment is described by S.Johnston et al., Science 240, 1538 (1988), and chloroplasttransformation in Chlamydomonas by particle bombardment is described byJ. Boynton et al., Science 240, 1534 (1988).

The use of particle bombardment for the transformation of animal tissueor cells has received comparatively little attention. Sanford et al.,Particulate Science and Technology 5, 27, 35-36 (1987), suggest the useof particle bombardment for human gene therapy, but do not suggest thetissue type or the developmental stage of tissue useful for carrying outsuch therapy. U.S. patent application Ser. No. 06/877,619, titled“Method for Transporting Substances Into Living Cells and Tissues andApparatus Therefor,” concerns the introduction of biological materialsinto cells by microprojectile bombardment. Suggested biologicalsubstances are stains such as fluorescent or radiolabled probes,viruses, organelles, vesicles, proteins such as enzymes or hormones, andnucleic acids such as DNA and RNA. Suggested procedures include: (a) theparticle bombardment of animal cells such as eggs, bone marrow cells,muscle cells, and epiderman cells at page 16, lines 5-6; (b) theparticle bombardment of human tissue or other animal tissue such asepidermal tissue, organ tissue, and tumor tissue at page 16, lines13-14; and (c) human gene therapy for sickle cell anemia by theparticle-mediated transformation of bone marrow tissue at page 22, lines8-9.

W. Brill, Particle Propulsion by Electric Discharge (Tape of Speech atAAAS meeting on Plant Molecular Biology/Genetic Engineering forAgriculture (VI) (January 1989), discusses the transformation ofnematodes to correct a missing body wall myosin gene by particlebombardment. The utility of transforming nematodes is, however,comparatively limited.

In view of the foregoing, an object of this invention is to provide newuses for the treatment of animals, particularly vertebrates, and theirtissues and cells, by microprojectile bombardment.

A more particular object of this invention is to use microprojectilebombardment as a means for administering proteins or peptides to ananimal subject.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of transferring agene to preselected vertebrate cells. The method comprises the steps of,first, providing microprojectiles, the microprojectiles carryingpolynucleic acid sequences, the sequences comprising, in the 5′ to 3′direction, a regulatory sequence operable in the vertebrate cells and aheterologous gene positioned downstream of the regulatory sequence andunder the transcriptional control thereof. The microprojectiles are thenaccelerated at the preselected cells, with the microprojectilescontacting the cells at a speed sufficient to penetrate the cells anddeposit the polynucleic acid sequences therein ( as used herein, theplural form of terms such as “cell,” “microparticle,” and “polynucleicacid sequence” is intended to encompass the singular).

A second aspect of the present invention is a method of transferring agene to preselected vertebrate tissue. The method comprises the stepsof, first, providing microprojectiles, the microprojectiles carryingpolynucleic acid sequences, the sequences comprising, in the 5′ to 3′direction, a regulatory sequence operable in the vertebrate tissue and aheterologous gene positioned downstream of the regulatory sequence andunder the transcriptional control thereof. The microprojectiles are thenaccelerated at the preselected tissue, with the microprojectilescontacting the cells of the tissue at a speed sufficient to penetratethe cells and deposit the polynucleic acid sequences therein.

A third aspect of the present invention is a method of transferring agene to a preselected tissue in situ in a vertebrate subject. The methodcomprises the steps of, first, providing microprojectiles, themicroprojectiles carrying polynucleic acid sequences, the sequencescomprising, in the 5′ to 3′ direction, a regulatory sequence operable inthe vertebrate tissue and a heterologous gene positioned downstream ofthe regulatory sequence and under the transcriptional control thereof.The microprojectiles are then accelerated at the animal subject, withthe subject positioned so that the microprojectiles contact thepreselected tissue, with the microprojectiles contacting the cells ofthe tissue at a speed sufficient to penetrate the cells and deposit thepolynucleic acid sequences therein.

The data disclosed herein provide the first demonstration ofparticle-mediated transformation of (a) vertebrate cells, (b) vertebratetissue, and (c) vertebrate tissue in situ of which these applicants areaware.

Also disclosed herein is a method of administering a protein or peptideto a vertebrate subject. This method is based in part on our findingthat vertebrate tissue transformed by particle bombardment issurprisingly free of callus formation, inflammation, and other defensiveresponses. Thus, proteins and peptides released from the transformedcells (by virtue of their being transformed) can circulate throughoutthe animal subject in which the cells reside, and cells which circulatein the animal subject (e.g., lymphocytes) have access to the transformedcells. In this method, target vertebrate tissue (preferably dermis orhypodermis tissue) is selected and microprojectiles provided. Themicroprojectiles carry polynucleic acid sequences, the sequencescomprising, in the 5′ to 3′ direction, a regulatory sequence operable inthe selected tissue and a gene positioned downstream of the regulatorysequence and under the transcriptional control thereof. The gene codesfor a protein or peptide. The microprojectiles are then accelerated atthe selected target tissue, with the microprojectiles contacting thecells of the tissue at a speed sufficient to penetrate the tissue cellsand deposit the polynucleic acid sequences therein to providetransformed tissue cells. The transformed tissue cells are thenmaintained in the animal subject, with the transformed tissue cellspresent in the subject in a number sufficient to produce a physiologicalresponse (e.g., an endocrine response, an immune response) to theprotein or peptide coded for by the gene in the subject upon expressionof the gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is explained in greater detail in the Examples,Detailed Description, and Figures herein, in which:

FIG. 1 is a perspective view of a currently available microprojectilebombardment apparatus;

FIG. 2 is a detailed view of the bombardment chamber shown in FIG. 1,with the stopping plate and animal chamber positioned for insertion;

FIG. 3 is a perspective view of an animal chamber positioned in abombardment chamber, with the animal chamber sealing plate positionedfor insertion;

FIG. 4 is a side sectional view of the apparatus shown in FIG. 3, andshowing the paths of travel of the macroprojectile to the stopping plateand the microprojectiles from the stopping plate to the subject;

FIG. 5 is a detailed view of a stopping plate and sealing plate, showingthe macroprojectile after impact on the sealing plate and the path oftravel of the microprojectiles to the sealing plate;

FIG. 6 shows the persistent heat-inducibility of the firefly luciferasegene driven by the human HSP70 promoter after transformation of culturedskeletal myotubes by microprojectile bombardment; and

FIG. 7 shows peak luciferase activity of skin and ear one day aftertransfection by microprojectile bombardment.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “tissue” means an aggregation of similarlyspecialized cells united in the performance of a particular function.The term “tissue cells” means cells residing in a tissue. The term“cell” means a cell either residing in a tissue (i.e., “in situ”) orremoved from its tissue of origin (i.e., “in vitro”).

Animal tissue cells can be bombarded in situ with respect to theirtissue of origin, or separated from the tissue and bombarded in vitro.The cells are preferably transformed in situ with respect to the tissueof origin. Tissue to be transformed can likewise be bombarded either invitro or in situ with respect to the animal of origin or the animal inwhich the transformed tissue is to subsequently be maintained, dependingon the result sought. Preferably, the tissue is transformed in situ inthe animal in which it is to be maintained.

Animal subjects to be treated by the method of the present invention arevertebrates, exemplary being fish, reptiles, amphibians, birds, andmammals. Birds (e.g., chicken, turkey) and mammals are preferred, andmammals (e.g., horse, cow, sheep, pig, human) are most preferred.Vertebrate tissues and cells to be treated by the method of the presentinvention are of corresponding origin, with the origins of the preferredand most preferred tissues and cells corresponding to the preferred andmost preferred animals.

The present invention may be practiced on any vertebrate cell, subjectto the proviso that cells which have been immortalized in cell cultureor otherwise altered from their native state are excluded. Thus, cellsto be transformed in the present invention are cells in their naturallyoccurring state (i.e., primary cells), whether they reside in a tissueor exist free from a tissue in vitro. Cells which have been maintainedin vitro for a time sufficient and/or under conditions effective tocause them to lose the characteristics they possess in situ are excludedfrom the group of cells with which the present invention is concerned.

Vertebrate cells to be treated by the method of the present inventionare preferably differentiated cells, and most preferably terminallydifferentiated cells such as skin cells, hypodermis cells, muscle cells,nerve cells, pancreas cells, and liver cells. Exemplary skin cellsinclude the basal cells and the cells of the dermis and hypodermis.

Vertebrate tissue to be treated by the method of the present inventionis likewise preferably differentiated tissue, and most preferablyterminally differentiated tissue such as skin tissue, hypodermis tissue,muscle tissue, nerve tissue, pancreas tissue, and liver tissue.Exemplary skin tissues include the basal cell layer, the dermis, and thehypodermis.

The polynucleic acid sequence carried by the microprojectile is arecombinant construct of a gene and a regulatory element. The constructmay take any suitable form, such as plasmid, a genomic viral DNAsequence such as a bovine papillomavirus vector, see E. Chen et al., 299Nature 529 (1982), a retroviral RNA sequence, derivatives of theforegoing, and synthetic oligonucleotides. The DNA constructs,particularly the plasmids, are currently preferred. Preferred geneswhich may be used in the polynucleic acid sequence are those which codefor a protein or peptide which produces a physiological response(preferably an endocrine response or an immune response) in the animalsubject. The gene may be homologous or heterologous with respect to theanimal to be transformed, or may be a modified version of a homologousgene.

Exemplary of genes which code for proteins or peptides which produce anendocrine response (i.e., a physiological response in the animal at apoint sufficiently removed from the transformed tissue region to requirethat the protein or peptide travel through the circulatory or lymphaticsystem of the subject) are genes which code for Factor VIII:C, geneswhich code for plasminogen activators such as Tissue PlasminogenActivator and urokinase, see, e.g., U.S. Pat. Nos. 4,370,417 and4,558,010, genes which code for growth hormones such as human or bovinegrowth hormone, genes which code for insulin, and genes which code forreleasing factors such as Luteinizing Hormone Releasing Hormone. Thedisclosures of all references cited herein are to be incorporated hereinby reference.

Exemplary of genes which code for proteins or peptides which produce animmune response (i.e., a response in which B and/or T lymphocytesactivated by the protein or peptide are capable of traveling in thecirculatory or lymphatic system of the subject to a site removed fromthe transformed tissue) are genes coding for subunit vaccines such asdisclosed in U.S. Pat. No. 4,857,634 to Minor et al. titled “PeptidesUseful in Vaccination against Enteroviruses,” U.S. Pat. No. 4,738,846 toRose et al. titled “Vaccine for Vesicular Stomatitis Virus,” U.S. Pat.No. 4,428,941 to Galibert et al. titled “Nucleotidic Sequence Coding theSurface Antigen of the Hepatitis B Virus, Vector Containing SaidNucleotidic Sequence, Process Allowing the Obtention Thereof and AntigenObtained Thereby,” and U.S. Pat. No. 4,761,372 to Maas et al. titled“Mutant Enterotoxin of E. coli.”

An advantage of administering a protein or peptide capable of producingan immune response in the manner described herein is the ability tocause the immunogen to be effectively presented to the subject over anextended period of time. This is in contrast to the simple injection ofa protein or peptide, which tend to be rapidly digested and cleared bythe subject.

Exemplary of other genes which code for proteins or peptides whichproduce a physiological response in the subject include genes coding forenzymes such as α₁ antitrypsin, genes which code for receptors such asthe insulin receptor, see U.S. Pat. No. 4,761,371 to Bell et al., geneswhich code for adhesons such as the CD4 receptor, see EPO PatentApplication Publication No. 0 314 317 of Genentech, titled “Adhesonvariants, nucleic acid encoding them and compositions comprising them,”which may have therapeutic activity in the subject, genes which code forproteins or peptides which will either affect neighboring tissue cells(a paracrine-like action) or will be secreted and affect the secretingcell (an autocrine-like action), and genes which code forpathogen-derived resistance. See J. Sanford and S. Johnston, 113 J.Theor. Biol. 395 (1985); J. Sanford, 130 J. Theor. Biol. 469 (1988).

The polynucleic acid sequence includes a regulatory sequence upstreamfrom, or 5′ to, the gene. The regulatory sequence is positioned in thepolynucleic acid sequence in operative association with the gene so asto be capable of inducing transcription of the gene. Regulatorysequences which may be used to provide transcriptional control of thegene in the polynucleic acid sequence are generally promoters which areoperable in the target tissue cells. Exemplary promoters include, forexample, the human α-actin promoter, see T. Miwa and L. Kedes, 7 Molec.Cell Biol. 2803 (1987), the human β-actin promoter, J. Leavitt et al., 4Molec. Cell Biol. 1961 (1984), the troponin T gene promoter, see T.Cooper and C. Ordahl, 260 J. Biol. Chem. 11140 (1985), the human heatshock protein (HSP) 70 promoter, retrovirus long terminal repeats suchas the Rous Sarcoma Virus long terminal repeat, see generally RNA TumorViruses (R. Weiss, N. Teich, H. Varmus and J. Coffin Eds. 2d ed. 1984),and the metallothionin gene promoter. The promoter and gene should becapable of operating in the cells, or cells of the tissue, to betransformed (i.e., the promoter should be capable of inductingtranscription of the gene, and the gene should code for an mRNA sequencecapable of being translated), with the requirements for operabilityknown in the art. See generally R. Old and S. Primrose, Principles ofGene Manipulation (3d Ed. 1985). With respect to tissue, these elementsneed only be operable in one cell type in that tissue.

Other regulatory elements which may optionally be incorporated into thepolynucleic acid sequence include enhancers, termination sequences, andpolyadenylation sites, as known in the art, as necessary to obtain thedesired degree of expression of the gene in the cell into which it isinserted.

Any microprojectile acceleration cell transformation apparatus can beused in practicing the present invention, so long as the apparatus ismodified as necessary for the treatment of air-breathing animals.Exemplary apparatus is disclosed in Sanford et al., Delivery ofSubstances into Cells and Tissues using a Particle Bombardment Process,5 Particulate Science and Technology 27 (1988), in Klein et al.,High-Velocity Microprojectiles for Delivering Nucleic Acids into LivingCells, 327 Nature 70 (1987), and in Agracetus European PatentApplication Publication No. 0 270 356, titled Pollen-Mediated Planttransformation. We used a commercially available device from Biolistics,Inc., 108 Langmuir Laboratory, Cornell Business and Technology Park,Brown Road, Ithaca, N.Y., 14850. This device is designated a Model BPG-4Particle Acceleration Apparatus and is configured essentially asdescribed in Klein et al., 327 Nature 70 (1987). The device, illustratedin FIGS. 1 through 5 (with improvements shown in FIGS. 2-5), comprises abombardment chamber 10 which is divided into two separate compartments11, 12 by an adjustable-height stopping plate support 13. Anacceleration tube 14 is mounted on top of the bombardment chamber. Amacroprojectile 15 is propelled down the acceleration tube at stoppingplate 16 by a gunpowder charge. A conventional firing mechanism 18 andevacuating apparatus 19 are provided. The stopping plate 16 has a borehole 17 formed therein which is smaller in diameter than themacroprojectile, the macroprojectile carries the microprojectiles, andthe macroprojectile is aimed and fired at the bore hole 17. When themacroprojectile 15 is stopped by the stopping plate 16, themicroprojectiles are propelled through the bore hole 17. The targettissue 40, here schematically illustrated as an animal subject, ispositioned in the bombardment chamber so that microprojectiles propelledthrough the bore hole 17 penetrate the cell membranes of the cells inthe target tissue and deposit DNA constructs carried thereon in thecells of the target tissue. The bombardment chamber 10 is partiallyevacuated prior to use to prevent atmospheric drag from unduly slowingthe microprojectiles. The chamber is only partially evacuated so thatthe target tissue is not unduly desiccated during bombardment thereof. Avacuum of between about 20 to 26 inches of mercury is suitable.

Microprojectiles (i.e., microparticles) used in carrying out the presentinvention may be formed from any material having sufficient density andcohesiveness to be propelled into the cells of the tissue beingtransformed, given the particle's velocity and the distance the particlemust travel. Non-limiting examples of materials for makingmicroprojectiles include metal, glass, silica, ice, polyethylene,polypropylene, polycarbonate, and carbon compounds (e.g., graphite,diamond). Metallic particles are currently preferred. Non-limitingexamples of suitable metals include tungsten, gold, and iridium. Theparticles should be of a size sufficiently small to avoid excessivedisruption of the cells they contact in the target tissue, andsufficiently large to provide the inertia required to penetrate to thecell of interest in the target tissue. Gold particles ranging indiameter from about one micrometer to about three micrometers arepreferred for in situ bombardment, and (more particularly) tungstenparticles about one micrometer in diameter are preferred for in vitrobombardment of muscle.

The polynucleic acid sequence may be immobilized on the particle byprecipitation. The precise precipitation parameters employed will varydepending upon factors such as the particle acceleration procedureemployed, as is known in the art. The carrier particles may optionallybe coated with an encapsulating agent such as polylysine to improve thestability of polynucleic acid constructs immobilized thereon, asdiscussed in EPO Application 0 270 356, at Column 8.

Skin in vertebrates is formed from an outer epidermis and an underlyingdermis (or corneum). Further underlying the dermis there usually is aloose, spongy layer called the hypodermis which is herein treated bydefinition as a part of the skin. The dermis and/or the hypodermis arethe preferred tissue targets when the object of the transformation is toadminister a protein or peptide to the animal subject in a manner whichwill evoke a physiological response thereto in the animal subject, asdiscussed above.

In land-dwelling vertebrates such as land-dwelling amphibians, reptiles,birds, and mammals, the epidermis is generally comprised of, from theouter surface to the inner surface, the following layers: (a) thestratum corneum, or horny layer, composed of thin squamous (flat)keratinized cells that are dead and continually being shed and replaced;(b) the stratum lucidum, or clear layer, in which keratinocytes areclosely packed and clear, and in which the nuclei are absent and thecell outlines indistinct; (c) the stratum granulosum, or granular celllayer, where the process of keratinization begins; (d) the stratumspinosum, or prickle cell layer, where cells are rich in ribonucleicacid and thereby equipped to initiate protein synthesis forkeratinization; and (e) the stratum basale, or basal cell layer, whichis composed of a single layer of columnar cells that are the only cellsin the epidermis that undergo mitosis. See generally G. Thibodeau,Anatomy and Physiology, 114-19 (1987); R. Frandson, Anatomy andPhysiology of Farm Animals, 205-12 (2d Ed. 1981); R. Nickel et al.,Anatomy of the Domestic Birds, 156-57 (1977).

The dermis, also called the “true skin,” is generally composed of astratum superficiale, or papillary layer, which immediately underliesthe epidermis, and an underlying stratum profundum, or reticular layer.The arteries, veins, capillaries, and lymphatics of the skin areconcentrated in the dermis. The reticular layer generally includes adense network of interlacing white collagenous fibers, skeletal muscles,and involuntary muscles. The papillary layer is composed of looseconnective tissue and a fine network of thin collagenous and elasticfibers.

The hypodermis, or superficial fascia, is a loose, spongy subcutaneouslayer rich in fat, areolar tissue, and blood vessels. When skin isremoved from an animal by blunt dissection, separation usually occurs inthe cleavage plane that exists between the hypodermis and underlyingtissues, with at least portions of the hypodermis thus adhering to theskin.

In the method of the present invention, dermis and epidermis may betransformed by either (a) propelling the microprojectiles through theepidermis, or (b) surgically exposing the hypodermis and dermis byincision and blunt dissection of a skin flap from the animal andpropelling the microprojectiles directly into the hypodermis and dermiswithout projecting the microprojectiles through the outer surface layer,and then restoring the dissected skin flap to the position on the animalfrom which it came. The skin flap can remain attached to the animal formicroprojectile bombardment or briefly removed for microprojectilebombardment and then grafted back to the animal. If removed from theanimal the skin flap can be returned to the same or a different site onthe animal, or can be transplanted to a different animal. We prefer toleave the skin flap attached. We have found greater transformation ofthe dermis by surgically exposing the dermis so that themicroprojectiles need not pass through the epidermis, but have alsofound substantial transformation of the dermis even when themicroprojectiles are propelled through the epidermis of land-dwellingvertebrates.

Various aspects of the present invention are explained in the exampleswhich follow. These examples are given to illustrate the invention, andare not to be construed as limiting thereof.

EXAMPLE 1 Particle-Mediated Transformation of Terminally DifferentiatedSkeletal Myotubes

This example demonstrates that primary cultures of fully differentiated,non-dividing skeletal myotubes can be transformed in vitro using aDNA-particle accelerator. The introduced genes are not rapidly degraded,but remain transcriptionally active over the life of the culture (twelvedays).

Myoblast cultures (4×10⁵ cells) were established from breast muscles ofeleven-day chick embryos in gelatin-coated 60 mm plastic dishes inDulbecco's Minimum Essential Medium (DMEM) supplemented with 10% horseserum and 5% embryo extract. After five days without fresh media,cultures consisted almost entirely of multinucleated myotubes, some ofwhich showed cross-striations. Some cultures were also treated withcytosine arabinoside (ara-C: 1.5 to 3.0 μg/ml) to inhibit growth ofresidual undifferentiated myoblasts or non-myogenic cells. See G.Paulath et al., Nature 337, 570 (1989). At this stage (five days inculture) the conditioned medium was removed and saved, and the plateswere placed in a vacuum chamber. Tungsten microprojectiles (meandiameter 1 μm) were coated with pHb-LUC, a plasmid construct in whichthe firefly luciferase gene, J. de Wet et al., Molec. Cell Biol. 7, 725(1987), is driven by the human B-actin promoter, J. Leavitt et al.,Molec. Cell Biol. 4, 1961 (1984), a promoter which has strongconstitutive activity in these cells. Each culture was bombarded undervacuum (twenty-nine inches Hg) with 2 μl of microprojectile suspension.The macroprojectile was started 3 cm from the top of the barrel andaccelerated with a #1 gunpowder 22 caliber cartridge. The petri dish wasplaced at the bottom of the chamber. The device used and the methods forcoating of the microprojectiles are described in J. Sanford et al.,Particulate Sci. Technol. 5, 27 (1987) and in T. Klein et al., Nature327, 70 (1987). In the present study, pilot experiments were performedto establish the particle velocity, particle size, particle composition,and cell density that resulted in maximal expression of luciferaseactivity following bombardment for our particular circumstances. Oncethese conditions were optimized, the experiments described in Table 1were performed. Whole cell lysates were prepared from cells two daysafter bombardment and luciferase activity was measured in a BertholdBiolumat LB9500C luminometer following addition of luciferin in thepresence of excess ATP. J. de Wet et al., Molec. Cell Biol. 7, 725(1987). These data are also shown in Table 1.

TABLE 1 Expression of firefly luciferase gene driven by the humanβ-actin promoter following transfection by microparticle bombardment offully differentiated skeletal myotubes. Luciferase activity (peak, lightemission/60 mm culture dish) Mock transfection 14 ± 7  (n = 3) pHB-LUC112,164 ± 19,086  (n = 6) pHB-LUC + ara-C 107,620 ± 19,881  (n = 6)

Transformation by microprojectiles produced reporter gene activitiesthat were 10-20× higher/plate and 200-400× higher/μg DNA than activitiesobtained by transformation of myotube cultures by standard calciumphosphate co-precipitation. See C. Chen and H. Okayama, Molec. CellBiol. 7, 2745 (1987).

EXAMPLE 2 Fate Over Time of DNA Introduced in Terminally DifferentiatedSkeletal Myotubules by Particle Bombardment

This Example addressed the fate of the introduced DNA over time bymeasuring the response of an inducible promoter at varying intervalsafter bombardment. Myotubes were transformed as described in Example 1above, but with the firefly luciferase gene under the control of thehuman HSP70 promoter. See B. Wu et al., Proc. Nat. Acad. Sci. USA 83,629 (1986). On days 2-7 following bombardment, luciferase activity wasmeasured in sister cultures that were either maintained at 37° C.(Control=C) or placed at 45° C. for 90 minutes followed by recovery at37° C. for three hours (Heat Shock=HS).

The cultures maintained for 6-7 days following bombardment were re-fedat two day intervals with conditioned media (depleted of mitogenicgrowth factors) from untransfected myotube cultures. FIG. 6 demonstratesthat inducible expression of the introduced plasmid was maintained overthis period. As calculated relative to the basal level expression in thecontrol plates, there was no diminution in expression between day 2(7.9-fold induction) and day 7 (11.9-fold induction) cultures. Thus,there was no substantial degradation of the plasmid or silencing of theheterologous promoter during the lifetime of these cultures.

EXAMPLE 3 Site of Transgene Expression in Cultures of TerminallyDifferentiated Myotubules

This Example was conducted to determine whether trans-gene expressionwas occurring within the fully differentiated myotubes, as distinguishedfrom mononuclear cells that remain within these primary cultures.Cultures of differentiated myotubes were transfected, by microprojectilebombardment as described in Example 1, with a plasmid constructcontaining the Drosophila Alcohol Dehydrogenase (ADH) gene under thecontrol of the Rous Sarcoma Virus long terminal repeat (pRSV-ADH). Onthe following day, cells were fixed and stained according to the methoddescribed by C. Ordahl et al., Molecular Biology of Muscle Development547 (C. Emerson et al. Eds. 1986), and photographed under phase contrastwith misaligned phase rings.

Although Drosophila ADH was detectable in mononuclear cells, the largemajority of the activity was found within multinucleated fused myotubes.Interestingly, two patterns of myotube staining were evident. In somemyotubes, ADH activity was spatially limited around a single nucleus,while the remainder of the multinucleated cell was devoid of activity.This staining pattern suggests that conditions existed in these cells torestrict expression of the transgene to a spatial domain surrounding anindividual nucleus. However, in other myotubes, ADH staining was;distributed uniformly through the cell. This diffuse pattern oftransgene expression implies either that multiple nuclei weretransformed in a single myotube, or that, under some conditions, the ADHprotein was free to diffuse throughout the entire span of theseelongated cells. In view of the apparent frequency of transformation,the latter explanation is favored.

EXAMPLE 4 Transformation of Alternate Cells with pRSV ADH

The experiment described in example 3 above was repeated in essentiallythe same manner, except that cardiac cells in vitro were used instead ofskeletal myotubes. No positive results were seen. The lack oftransformation was apparently due to the very few number of cells on theplate of cells used.

The experiment described in example 3 above was again repeated inessentially the same manner, except that whole mouse diaphragm was usedinstead of skeletal myotubes. The diaphragm was held flat on a dish witha piece of screen and the screen held down by weights. No transformationwas seen. It appears that the 1 micron tungsten microprojectilesemployed did not have sufficient kinetic energy to penetrate thediaphragm tissue.

EXAMPLE 5 Apparatus for Transformation of Animals

The device employed in the above examples were modified for thetransformation of tissue in whole animals in the manner illustrated inFIGS. 1 through 5. The door 20 on the bombardment chamber 10 was openedand an animal bombardment fitting, 30 or “trap,” inserted. The animalbombardment fitting included a cover plate 31 and an animal chamber 32.The animal chamber 32 has a top wall, bottom wall, side walls, a backwall, and an outer flange 33. The chamber 32 is inserted through anopening in the cover plate and sealed thereto by means of a rubbergasket on the front side of the cover plate positioned between the coverplate opening and the edge of the animal chamber flange. Threadedfasteners 34 secure the animal chamber 32 to the cover plate 31. Theback side of the cover plate has a rubber gasket spaced inwardly fromthe outer edge thereof for sealing the cover plate to the bombardmentchamber 10.

The top of the animal chamber has an opening 35 formed therein which,when the animal chamber is installed in the bombardment chamber, isaxially aligned with the center axis of the bore hole 17 of the stoppingplate 16. An inner cylindrical sleeve 36, open at the top and bottom, isconnected and sealed in the top opening 35. The bottom edge portion ofthe inner sleeve has a rubber gasket 37 inserted therein.

A sealing plate 38 is provided for sealing the bottom opening of theinner sleeve 36. The sealing plate has a center opening 39 formedtherein. The surface of the tissue on the animal subject 40 to betransformed is placed in contact with the sealing plate 38 so that thetissue to be transformed is accessible through the center opening 39.The sealing plate 39 is then placed in contact with the bottom edgeportion of the inner sleeve 36 and a vacuum drawn in the vacuum chamber.The contact of the subject tissue to the sealing plate 38, the sealingplate to the inner sleeve 36, the inner sleeve to the animal chamber 32,the animal chamber to the cover plate 31 and the cover plate to thebombardment chamber 10 all operate to seal the bombardment chamber 10. Ascreen is provided across the center opening of the sealing plate on thebottom surface thereof to reduce the tendency of tissue to be drawn intothe chamber. When the microprojectiles are accelerated, the opening 39in the sealing plate 38 is positioned so that the microprojectilescontact the tissue surface accessible through the opening. A sponge orother spacing means 41 can be used to hold the animal subject up againstthe sealing plate.

EXAMPLE 6 Particle Bombardment of Euthanized Mice

Mice were euthanized and the hair removed from their hind legs with adepilatory (NEET™) to expose the skin on the hind legs. The skin wasthen either left in position or dissected away to expose underlyingmuscle. The animals were positioned in the apparatus described inexample 5 above, either hind leg skin or muscle tissue positioned forbombardment, a vacuum of 26 inches of mercury drawn in the chamber, andthe tissue bombarded with 1 micron tungsten microprojectiles. 1 microntungsten particles were found too small to penetrate either muscle orskin. 3.4 micron tungsten particles were then tried, and were found topenetrate muscle and skin. Best were gold particles, 1 to 3 microns indiameter.

EXAMPLE 7 Particle Bombardment of Skin and Ear in Live Mice

Live adult female Balb C and Charles River CD1 mice were transformed bythe apparatus and procedures described in the preceding examples. Goldparticles 1 to 3 microns in diameter were coated with pHb-LUC byprecipitation, as described above. Animals were anesthetized with amixture containing equal parts of ketamine and xylazine (0.067 mg/g bodyweight). The target areas were hind leg skin and ear, which wereprepared with a depilatory as described above. A vacuum of 26 inches ofmercury was drawn for hind leg skin and 20 inches of mercury drawn forear. After bombardment, the tissue showed little or no evidence ofdamage. A faint brown stain was evident in the area containing particlesin most animals and, rarely, a small (<1 mm²) area of intradermalhemorrhage from small blood vessels was noted. Peak luciferase activityof the skin and ears on day one after transfection in counts per minuteis shown in FIG. 7. The values are means ±the standard deviation. Theactivity of 17 skin samples was 4,699±4,126 and the activity of 12 earsamples was 47,114±3,679. Photoluminescence was determined in duplicateon a Berthold LB 9500 C luminometer set for a ten second period ofintegration with 50 microliter (skin) and 25 microliter (ear) samples ofextract. The mean luciferase activity for skin and ear over time incounts per minute is shown in Table 2 below.

TABLE 2 Luciferase Activities for Days 1 to 4 DAY: 1 2 3 4 Skin: mean 4699   810  217 104 s.d.  4126  1097  265 140 Ear: mean 149369 11611469986 s.d.  49392 122455 66461

After recovery from anesthesia, animals showed no behavioralabnormalities and did not manifest evidence of pain or itching in thetransformed area of skin. Histologic examination of bombarded skinrevealed no significant alteration of tissue structure, and onlyoccasional lymphocytes or polymorphonuclear leukocytes within thetransformed area. In situ hybridization studies revealed a highproportion (approximately 25%) of cells within the epidermis thatexpressed luciferase mRNA, and a lower (but noticable) proportion in thedermis had hair follicles.

EXAMPLE 8 Local Transgene Activity in Ear of Live Mice by ParticleBombardment

Mouse ears were transformed with pGH precipitated on 1 to 3 micron goldmicroparticles as described in Example 7 above. The plasmid pGH includesa human growth hormone (HGH) gene driven by a metallothionin promoter.Local levels of HGH were measured with a commercially available NicholsInstitute Allegro™ HGH Radioimmunoassay. The RIA data is given in Table3 below. Activity is expressed in counts per minute.

TABLE 3 Local HGH Activity in Mouse Ear Group Activity Positive Control441 Left Ear 520 Right Ear 220 Negative Control  90 Negative Control  67

The foregoing examples are illustrative of the present invention, andare not to be taken as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of administering a protectiveimmune response-producing protein or peptide to produce a protectiveimmune response in a vertebrate subject by in situ microprojectilebombardment, said method comprising the steps of: (a) selecting a targetskin tissue residing in a vertebrate subject, the tissue selected fromthe group consisting of epidermis tissue, dermis tissue, and hypodermistissue; (b) providing microprojectiles, the microprojectiles carrying aDNA sequence comprising, in the 5′ to 3′ direction, a regulatory elementfunctional in said target skin tissue and a gene positioned downstreamof the regulatory element and under transcriptional control thereof toexpress said gene, the gene coding for a protective immuneresponse-producing protein or polypeptide, wherein the microprojectilescomprise a material selected from the group consisting of metal, glass,silica, ice, polyethylene, polypropylene, polycarbonate, graphite anddiamond; (c) accelerating the microprojectiles at the subject, with thesubject positioned so that the microprojectiles contact the subject'sepidermis at a speed sufficient to penetrate the epidermis and lodge insaid selected target skin tissue cells and deposit the DNA sequencetherein to provide transformed skin tissue cells, whereby a protectiveimmune response-producing protein is administered to the vertebratesubject; and (d) maintaining the transformed skin tissue cells in thesubject, wherein a protective immune response is produced to the proteinor peptide coded for by said gene in the subject upon expression of saidgene in said transformed skin tissue cells, the immune responsecomprising a response in which B lymphocytes, T lymphocytes, or both Band T lymphocytes are activated by the protein or peptide and travel incirculatory or lymphatic systems of the vertebrate subject to a siteremoved from the transformed skin tissue cells.
 2. A method according toclaim 1, wherein the transformed skin tissue cells increase the systemicconcentration of the protein or peptide coded for by said gene in thesubject upon expression of said gene in said transformed skin tissuecells.
 3. A method according to claim 1, wherein the gene comprises aheterologous gene coding for an immune response-producing protein orpolypeptide with respect to the vertebrate subject.
 4. A methodaccording to claim 1, wherein said gene codes for growth hormone.
 5. Amethod according to claim 1, wherein the microprojectiles are propelledthrough the epidermis of the subject.
 6. A method according to claim 1,wherein said microprojectiles have diameters between 1 micron and 3microns.
 7. A method according to claim 1, wherein said microprojectileis formed from metal.
 8. A method according to claim 1, wherein saidmicroprojectile is formed from a metal selected from the groupconsisting of tungsten, gold, and iridium.
 9. A method of administeringa protective immune response-producing protein or peptide to produce aprotective immune response in a vertebrate subject by in situmicroprojectile bombardment, said method comprising: (a) selecting atarget skin tissue residing in a vertebrate subject, the tissue selectedfrom the group consisting of epidermis tissue, dermis tissue, andhypodermis tissue; (b) providing microprojectiles, the microprojectilescontacting with isolated and purified DNA constructs comprising, in the5′ to 3′ direction, a regulatory element functional in said target skintissue and a gene positioned downstream of the regulatory element andunder transcriptional control thereof to express said gene, the genecoding for a protective immune response-producing protein orpolypeptide, wherein the microprojectiles comprise a material selectedfrom the group consisting of metal, glass, silica, ice, polyethylene,polypropylene, polycarbonate, graphite and diamond; (c) accelerating themicroprojectiles at the subject, with the subject positioned so that themicroprojectiles contact the subject's epidermis at a speed sufficientto penetrate the epidermis and lodge in said selected target skin tissuecells and deposit the DNA constructs therein to provide transformed skintissue cells, whereby a protective immune response-producing protein isadministered to the vertebrate subject; and (d) maintaining thetransformed skin tissue cells in the subject, wherein a protectiveimmune response is produced to the protein or peptide coded for by saidgene in the subject upon expression of said gene in said transformedskin tissue cells, the immune response comprising a response in which Blymphocytes, T lymphocytes, or both B and T lymphocytes are activated bythe protein or peptide and travel in circulatory or lymphatic systems ofthe vertebrate subject to a site removed from the transformed skintissue cells.
 10. A method according to claim 9, wherein themicroprojectiles have a diameter ranging from about one to about threemicrometers.
 11. A method according to claim 9, wherein the metal isselected from the group consisting of tungsten, gold and iridium.